Posttranscriptionally Mediated Decreases in Folylpolyglutamate Synthetase Gene Expression in Some Folate Analogue-resistant Variants of the L1210 Cell

Abstract

L1210 cell variants resistant to edatrexate (EDX) were isolated by selection in vivo during therapy with this folate analogue. Among the variants selected, seven (L1210/EDX-4 to -7 and L1210/EDX-12 to -14) were found to exhibit 2-23-fold lower levels of folylpolyglutamate synthetase (FPGS) activity compared with parental L1210 cells. Lower levels of FPGS activity in cell-free extract from these variants using EDX as substrate were characterized by the same relative decrease in value for Biochemical alterations associated with acquired resistance of tumor cells to classical folate analogues are highly diverse (1-5). This phenotypic diversity most likely reflects the common occurrence of multiple genomic alterations among clonal variants selected for resistance to these agents either in vitro or in vivo. As a consequence, acquired resistance to methotrexate and newer folate analogues under development will remain (6 -8) a major limitation to their effective clinical utility. One of the determinants of cytotoxicity shared (9 -11) by most folate analogues, whether targeted to dihydrofolate reductase or folatedependent biosynthetic enzymes, is the process of intracellular polyglutamylation mediated by the enzyme folylpolyglutamate synthetase (FPGS).1 This process is not only important to the conservation and efficient utilization by proliferating cells of folate coenzymes (12-18) but is responsible, as well, for converting folate analogues (9 -18) to longer chain polyglutamate anabolites that are more retentive and more effective inhibitors of folate-dependent enzymatic targets. Subsequently, examples have been reported from our laboratory (4, 19) and elsewhere (20, 21) of tumor cell variants with acquired resistance to methotrexate and other folate analogues that exhibit lower levels of FPGS activity when compared with parental cells and are cross-resistant to most other classical folate analogues. In one of our own studies with the L1210 leukemia, resistant variants (L1210/EDX) were independently selected (4) during therapy of tumor-bearing mice with edatrexate (EDX). Some of these variants exhibited levels of FPGS activity that were substantially lower than in parental cells. In the current studies, we address the underlying basis for these alterations in FPGS activity in these resistant variants at the level of the FPGS protein elaborated by these cells as well as cognate gene expression. Our results appear to define a novel mechanism underlying these modifications in FPGS activity in these variants that may have broad significance in regard to the regulation of FPGS gene expression and for therapy with classical folate analogues. A preliminary report of some of these findings has already been presented (22) in abstract form. EXPERIMENTAL PROCEDURESCells and Culture Conditions-Methods for the isolation of variants of the L1210 cell used in these studies were described in detail earlier (4). These variants were independently isolated from ascite fluid in the peritoneal cavity of mice following treatment with EDX. ...

Aberrant splicing of folylpolyglutamate synthetase as a novel mechanism of antifolate resistance in leukemia

“…Consistently, the mechanism that was identified in edatrexate-resistant L1210 leukemia cells was decreased rate of FPGS translation as judged from the poor translatability of the FPGS transcript in a cell-free translation system. 30 In the current study, we discovered another posttranscriptional alteration involving splicing defects in FPGS mRNA leading to loss of FPGS activity. However, whereas quantitative RT-PCR analysis, particularly with nondiagnostic PCR fragments, may frequently fail to identify such splicing defects, 14 Northern blot analysis proved instrumental in identification of changes in the full-length FPGS transcripts suggesting putative aberrant splicing ( Figure 4A); this was corroborated with diagnostic PCR analysis.…”

“…Moreover, the latter further offers a novel molecular basis for the frequent emergence of tumor cells with antifolate-resistant phenotypes that display apparently normal FPGS mRNA levels commonly evaluated by RT-PCR analysis, while exhibiting loss of FPGS activity in the absence of inactivating point mutations. 5,10,13,14,17 In this respect, it has been frequently shown that loss of FPGS activity occurs in the absence of a parallel decrease in mRNA levels in multiple CCRF-CEM leukemia cells with acquired resistance to various polyglutamatable antifolates, 10,13,14,17,20 including MTX, 10,17,29 raltirexed, edatrexate, 30 or lometrexol (DDATHF). 13 This finding strongly suggested the existence of alterations at the posttranscriptional level of the FPGS gene.…”

A Mouse Gene That Coordinates Epigenetic Controls and Transcriptional Interference To Achieve Tissue-Specific Expression

“…In striking contrast, the CpG island surrounding P2 was completely unmethylated in all tissues studied, including normal and neoplastic tissues in which the P2 promoter was transcriptionally active (L1210 cells and marrow); liver, in which transcripts initiating at P2 were barely detectable; and brain, in which the entire fpgs gene was transcriptionally inactive (Table 1). Interestingly, the P2 promoter showed an unmethylated state starting from the position previously found to contain transcription factor binding sites controlling this promoter (17,61) and continuing until just before the second exon. We note that, over the differentially methylated P1, H3 and H4 acetylation correlates with transcriptional activity but, over the unmethylated CpG island-P2 promoter region, H3 and H4 acetylation and transcriptional activity were independent.…”

From methotrexate to pemetrexed and beyond. A review of the pharmacodynamic and clinical properties of antifolates

“…Table 1 [1][2][3][4][5][6][7] provides a summary of the key molecular features and clinical development status of the drugs reviewed.…”

“…G3/4 toxicity (% of patients): neutropenia (21) increased transaminases(14), neurotoxicity (7), diarrhea (7), stomatitis (2), emesis(2).…”